Although several early phase clinical trials raised enthusiasm for the use of insulin-like growth factor I receptor (IGF1R)-specific antibodies for cancer treatment, initial Phase III results in unselected patients have been disappointing. Further clinical studies may benefit from the use of predictive biomarkers to identify probable responders, the use of rational combination therapies and the consideration of alternative targeting strategies, such as ligand-specific antibodies and receptor-specific tyrosine kinase inhibitors. Targeting insulin and IGF signalling also needs to be considered in the broader context of the pathophysiology that relates obesity and diabetes to neoplasia, and the effects of anti-diabetic drugs, including metformin, on cancer risk and prognosis. The insulin and IGFI receptor family is also relevant to the development of PI3K-AKT pathway inhibitors.

Rather than being a passive, haphazard process of wear and tear, lifespan can be modulated actively by components of the insulin/insulin-like growth factor I (IGFI) pathway in laboratory animals. Complete or partial loss-of-function mutations in genes encoding components of the insulin/IGFI pathway result in extension of life span in yeasts, worms, flies, and mice. This remarkable conservation throughout evolution suggests that altered signaling in this pathway may also influence human lifespan. On the other hand, evolutionary tradeoffs predict that the laboratory findings may not be relevant to human populations, because of the high fitness cost during early life. Here, we studied the biochemical, phenotypic, and genetic variations in a cohort of Ashkenazi Jewish centenarians, their offspring, and offspring-matched controls and demonstrated a gender-specific increase in serum IGFI associated with a smaller stature in female offspring of centenarians. Sequence analysis of the IGF1 and IGF1 receptor (IGF1R) genes of female centenarians showed overrepresentation of heterozygous mutations in the IGF1R gene among centenarians relative to controls that are associated with high serum IGFI levels and reduced activity of the IGFIR as measured in transformed lymphocytes. Thus, genetic alterations in the human IGF1R that result in altered IGF signaling pathway confer an increase in susceptibility to human longevity, suggesting a role of this pathway in modulation of human lifespan.

Although some cancers are initially sensitive to EGFR tyrosine kinase inhibitors (TKIs), resistance invariably develops. We investigated mechanisms of acquired resistance to the EGFR TKI gefitinib by generating gefitinib-resistant (GR) A431 squamous cancer cells. In GR cells, gefitinib reduced phosphorylation of EGFR, ErbB-3, and Erk but not Akt. These cells also showed hyperphosphorylation of the IGFI receptor (IGFIR) and constitutive association of IRS-1 with PI3K. Inhibition of IGFIR signaling disrupted the association of IRS-1 with PI3K and restored the ability of gefitinib to downregulate PI3K/Akt signaling and to inhibit GR cell growth. Gene expression analyses revealed that GR cells exhibited markedly reduced IGF-binding protein 3 (IGFBP-3) and IGFBP-4 RNA. Addition of recombinant IGFBP-3 restored the ability of gefitinib to downregulate PI3K/Akt signaling and to inhibit cell growth. Finally, gefitinib treatment of mice with A431 xenografts in combination with an IGFIR-specific monoclonal antibody prevented tumor recurrence, whereas each drug given alone was unable to do so. These data suggest that loss of expression of IGFBPs in tumor cells treated with EGFR TKIs derepresses IGFIR signaling, which in turn mediates resistance to EGFR antagonists. Moreover, combined therapeutic inhibition of EGFR and IGFIR may abrogate this acquired mechanism of drug resistance and is thus worthy of prospective clinical investigation.

Exposure of cells to a variety of stresses induces compensatory activations of multiple intracellular signaling pathways. These activations can play critical roles in controlling cell survival and repopulation effects in a stress-specific and cell type-dependent manner. Some stress-induced signaling pathways are those normally activated by mitogens such as the EGFR/RAS/PI3K-MAPK pathway. Other pathways activated by stresses such as ionizing radiation include those downstream of death receptors, including pro-caspases and the transcription factor NFKB. This review will attempt to describe some of the complex network of signals induced by ionizing radiation and other cellular stresses in animal cells, with particular attention to signaling by growth factor and death receptors. This includes radiation-induced signaling via the EGFR and IGFI-R to the PI3K, MAPK, JNK, and p38 pathways as well as FAS-R and TNF-R signaling to pro-caspases and NFKB. The roles of autocrine ligands in the responses of cells and bystander cells to radiation and cellular stresses will also be discussed. Based on the data currently available, it appears that radiation can simultaneously activate multiple signaling pathways in cells. Reactive oxygen and nitrogen species may play an important role in this process by inhibiting protein tyrosine phosphatase activity. The ability of radiation to activate signaling pathways may depend on the expression of growth factor receptors, autocrine factors, RAS mutation, and PTEN expression. In other words, just because pathway X is activated by radiation in one cell type does not mean that pathway X will be activated in a different cell type. Radiation-induced signaling through growth factor receptors such as the EGFR may provide radioprotective signals through multiple downstream pathways. In some cell types, enhanced basal signaling by proto-oncogenes such as RAS may provide a radioprotective signal. In many cell types, this may be through PI3K, in others potentially by NFKB or MAPK. Receptor signaling is often dependent on autocrine factors, and synthesis of autocrine factors will have an impact on the amount of radiation-induced pathway activity. For example, cells expressing TGFalpha and HB-EGF will generate protection primarily through EGFR. Heregulin and neuregulins will generate protective signals through ERBB4/ERBB3. The impact on radiation-induced signaling of other autocrine and paracrine ligands such as TGFbeta and interleukin 6 is likely to be as complicated as described above for the ERBB receptors.

Activation of the maternal immune system in rodent models sets in motion a cascade of molecular pathways that ultimately result in autism- and schizophrenia-related behaviors in offspring. The finding that interleukin-6 (IL-6) is a crucial mediator of these effects led us to examine the mechanism by which this cytokine influences fetal development in vivo. Here we focus on the placenta as the site of direct interaction between mother and fetus and as a principal modulator of fetal development. We find that maternal immune activation (MIA) with a viral mimic, synthetic double-stranded RNA (poly(I:C)), increases IL-6 mRNA as well as maternally-derived IL-6 protein in the placenta. Placentas from MIA mothers exhibit increases in CD69+ decidual macrophages, granulocytes and uterine NK cells, indicating elevated early immune activation. Maternally-derived IL-6 mediates activation of the JAK/STAT3 pathway specifically in the spongiotrophoblast layer of the placenta, which results in expression of acute phase genes. Importantly, this parallels an IL-6-dependent disruption of the growth hormone-insulin-like growth factor (GH-IGF) axis that is characterized by decreased GH, IGFI and IGFBP3 levels. In addition, we observe an IL-6-dependent induction in pro-lactin-like protein-K (PLP-K) expression as well as MIA-related alterations in other placental endocrine factors. Together, these IL-6-mediated effects of MIA on the placenta represent an indirect mechanism by which MIA can alter fetal development.

Caloric restriction (CR), reduced protein, methionine, or tryptophan diets; and reduced insulin and/or IGFI intracellular signaling can extend mean and/or maximum lifespan and delay deleterious age-related physiological changes in animals. Mice and flies can shift readily between the control and CR physiological states, even at older ages. Many health benefits are induced by even brief periods of CR in flies, rodents, monkeys, and humans. In humans and nonhuman primates, CR produces most of the physiologic, hematologic, hormonal, and biochemical changes it produces in other animals. In primates, CR provides protection from type 2 diabetes, cardiovascular and cerebral vascular diseases, immunological decline, malignancy, hepatotoxicity, liver fibrosis and failure, sarcopenia, inflammation, and DNA damage. It also enhances muscle mitochondrial biogenesis, affords neuroprotection; and extends mean and maximum lifespan. CR rapidly induces antineoplastic effects in mice. Most claims of lifespan extension in rodents by drugs or nutrients are confounded by CR effects. Transcription factors and co-activators involved in the regulation of mitochondrial biogenesis and energy metabolism, including SirT1, PGC-1alpha, AMPK and TOR may be involved in the lifespan effects of CR. Paradoxically, low body weight in middle aged and elderly humans is associated with increased mortality. Thus, enhancement of human longevity may require pharmaceutical interventions.

The early growth response 1 (Egr1) gene is a transcription factor that acts as both a tumor suppressor and a tumor promoter. Egr1-null mouse embryo fibroblasts bypass replicative senescence and exhibit a loss of DNA damage response and an apparent immortal growth, suggesting loss of p53 functions. Stringent expression analysis revealed 266 transcripts with >2-fold differential expression in Egr1-null mouse embryo fibroblasts, including 143 known genes. Of the 143 genes, program-assisted searching revealed 66 informative genes linked to Egr1. All 66 genes could be placed on a single regulatory network consisting of three branch points of known Egr1 target genes: TGFbeta1, IL6, and IGFI. Moreover, 19 additional genes that are known targets of p53 were identified, indicating that p53 is a fourth branch point. Electrophoretic mobility shift assay as well as chromatin immunoprecipitation confirmed that p53 is a direct target of Egr1. Because deficient p53 expression causes tumors in mice, we tested the role of Egr1 in a two-step skin carcinogenesis study (144 mice) that revealed a uniformly accelerated development of skin tumors in Egr1-null mice (P < 0.005). These studies reveal a new role for Egr1 as an in vivo tumor suppressor.

Insulin elicits a spectrum of biological responses by binding to its cell surface receptor. In a screen for small molecules that activate the human insulin receptor tyrosine kinase, a nonpeptidyl fungal metabolite (L-783,281) was identified that acted as an insulin mimetic in several biochemical and cellular assays. The compound was selective for insulin receptor versus insulin-like growth factor I (IGFI) receptor and other receptor tyrosine kinases. Oral administration of L-783,281 to two mouse models of diabetes resulted in significant lowering in blood glucose levels. These results demonstrate the feasibility of discovering novel insulin receptor activators that may lead to new therapies for diabetes.

The 14-3-3 proteins have been implicated as potential regulators of diverse signaling pathways. Here, using two-hybrid assays and in vitro assays of protein interaction, we show that the epsilon isoform of 14-3-3 interacts with the insulin-like growth factor I receptor (IGFIR) and with insulin receptor substrate I (IRS-1), but not with the insulin receptor (IR). Coprecipitation studies demonstrated an IGFI-dependent in vitro interaction between 14-3-3-glutathione S-transferase proteins and the IGFIR. In similar studies no interaction of 14-3-3 with the IR was observed. We present evidence to suggest that 14-3-3 interacts with phosphoserine residues within the COOH terminus of the IGFIR. Specifically, peptide competition studies combined with mutational analysis suggested that the 14-3-3 interaction was dependent upon phosphorylation of IGFIR serine residues 1272 and/or 1283, a region which has been implicated in IGFIR-dependent transformation. Phosphorylation of these serines appears to be dependent upon prior IGFIR activation since no interaction of 14-3-3 was observed with a kinase-inactive IGFIR in the two-hybrid assay nor was any in vitro interaction with unstimulated IGFIR derived from mammalian cells. We show that the interaction of 14-3-3 with IRS-1 also appears to be phosphoserine-dependent. Interestingly, 14-3-3 appears to interact with IRS-1 before and after hormonal stimulation. In summary, our data suggest that 14-3-3 interacts with phosphoserine residues within the COOH terminus of the IGFIR and within the central domain of IRS-1. The potential functional roles which 14-3-3 may play in IGFIR and IRS-1-mediated signaling remain to be elucidated.

The EGF-like homeotic gene Dlk1 appears to function as an inhibitor of adipogenesis. Overexpression of Dlk1 prevents adipogenesis of 3T3-L1 cells. Dlk1-deficient mice are obese; however, adipose tissue still develops in Fc-dlk1 transgenic mice, suggesting that Dlk1 is not a strict inhibitor of adipogenesis. To clarify the role of Dlk1 in adipogenesis, we studied whether Dlk1 could act differently on this process depending upon the differentiation state of the precursor cells. We found that Dlk1 is a potentiator of adipogenesis for mesenchymal C3H10T1/2 cells. This potentiating effect can be triggered by overexpressing the entire protein or the extracellular EGF-like-containing region, but not by overexpressing the intracellular dlk1 sequence. In addition, coculture of C3H10T1/2 cells with other cells expressing Dlk1, but not with cells lacking Dlk1 expression, enhances their adipogenic response. Potentiation of adipogenesis by Dlk1 was associated with changes in the activation of ERK1/2 after IGFI/insulin induction. Finally, as reported with other cells, dlk1 functioned as a Notch signaling inhibitor in C3H10T1/2 cells, but inhibition of Notch1 expression prevented the potentiating effects of Dlk1 in adipogenesis. These data suggest that Dlk1 may potentiate or inhibit adipogenesis depending upon the cellular context, and that Notch1 expression and activation are important factors in this context.

Insulin-like growth factors bind with high affinity to specific binding proteins in extracellular fluids. To identify structural characteristics of IGF-binding proteins that might define their physiological roles, we determined the complete primary structure of a novel human IGF-binding protein (IGFBP-2) from a cloned cDNA. The cDNA encodes a 328 amino acid IGF-binding protein precursor which contains a 39-residue signal peptide. The mature 289 amino acid IGFBP-2 has a predicted Mr of 31,325. Chinese hamster ovary (CHO) cells stably transformed with the IGFBP-2 cDNA secreted a 36 kd protein which bound, with different affinities, IGFII and IGFI, but did not bind insulin. The predicted protein sequence of this IGF-binding protein shares extensive amino acid homology (greater than 85%) with the IGF-binding protein secreted by rat BRL-3A cells, but less than 40% homology with human IGFBP-1. Therefore IGFBP-2, and not IGFBP-1 as previously suggested, represents the human homologue of the rat BRL-BP (alpha IGFBP-2). Moreover, from alignment of the predicted protein sequences of IGFBP-1 and IGFBP-2, extensive conservation of the distribution of cysteine residues is observed. Although the overall amino acid homology shared by these proteins is not high, we suggest that they represent a family of structurally related human IGFBPs. Southern blot analysis of human DNA demonstrates that IGFBP-2 is encoded by a single-copy gene, different from that of IGFBP-1.

Sorafenib is the first-line treatment for advanced hepatocellular carcinoma (HCC), but the clinical response to sorafenib is seriously limited by drug resistance. In this study, we investigated the molecular mechanisms of sorafenib resistance in HCC cells. Our miRNA microarray data indicate that liver-specific miR-122 expression was significantly reduced in sorafenib-resistant cells. Overexpression of miR-122 made drug-tolerant cells sensitive to sorafenib and induced apoptosis. Insulin-like growth factor 1 receptor (IGF-1R) was validated as a target of miR-122 and was repressed by this miRNA. miR-122-induced apoptosis was repressed by the IGF-1R activator IGFI or IGFII. Conversely, the IGF-1R inhibitor PPP or NVP-AEW541 in combination with sorafenib significantly induced cell apoptosis and disrupted tolerance to drugs in vitro. These results indicated that activation of IGF-1R by ectopic down-regulation of miR-122 counteracted the effects of sorafenib-induced apoptosis, thus conferring sorafenib resistance. Further study revealed that activation of IGF-1R by miR-122 down-regulation contributed to activation of RAS/RAF/ERK signaling, which was associated with drug resistance. Our data imply that an intimate correlation between miR-122 and IGF-1R abnormal expression is a critical determinant of sorafenib tolerance.

Fish endure long periods of fasting and demonstrate an extensive capacity for rapid and complete recovery after refeeding. The underlying mechanisms through which nutrient intake activates an increase in somatic growth and especially in muscle growth is poorly understood. In this study we examined the expression profile of major muscle growth regulators in trout white muscle 4, 12, and 34 days after refeeding, using real-time quantitative RT-PCR. Mean insulin-like growth factor I (IGFI) mRNA level in muscle increased dramatically 8- and 15-fold, 4 and 12 days, respectively, after refeeding compared to fasted trout. This declined thereafter. Conversely, only a weak but gradual increase in mean insulin-like growth factor II (IGFII) mRNA level was observed during refeeding. Inversely to IGFI, mean IGF receptor Ia (IGFRIa) mRNA level declined after ingestion of food. In contrast, IGF receptor Ib (IGFRIb) mRNA level was not affected by refeeding. Mean fibroblast growth factor 2 (FGF2) mRNA level increased by 2.5-fold both 4 and 12 days after refeeding, whereas fibroblast growth factor 6 (FGF6) and myostatin mRNA levels were unchanged. Subsequent to IGFI and FGF2 gene activation, an increase in myogenin mRNA accumulation was observed at 12 days post-refeeding suggesting that an active differentiation of myogenic cells succeeds their proliferation. In conclusion, among the potential growth factors we examined in this study, IGFI and FGF2 were identified as candidate genes whose expression may contribute to muscle compensatory growth induced by refeeding.

OBJECTIVE

To unravel the role of interleukin (IL)-6 and insulin-like growth factor (IGF)-I receptor (IGFIR) in expressing stemness-related properties and to evaluate the prognostic values of pluripotent transcription factor OCT4/NANOG, and IGFIR in hepatocellular carcinoma (HCC).

METHODS

Serum levels of IL6 were detected using ELISA assays (n = 120). The effects of IL6/IGFI on stemness expression in HCC were examined using OCT4/NANOG promoter luciferase reporter, RNA interference, secondary sphere formation, side population, and xenograft animal models. The OCT4/NANOG protein and phospho-IGFI receptor (p-IGFIR) in tissues were detected by Western blotting (n = 8) and immunohistochemical staining (n = 85). OCT4, NANOG, and IGFIR expression levels in tissues (n = 191) were analyzed by real-time qRT-PCR and was correlated with early tumor recurrence using the Kaplan-Meier survival analysis.

RESULTS

A high positive correlation between the expression levels of OCT4/NANOG and IGFIR/p-IGFIR in human HCC tissues was observed. The concurrent expression of OCT4/NANOG/IGFIR was mostly confined to hepatitis B virus (HBV)-related HCC (HBV-HCC) and was significantly correlated with early tumor recurrence. High serum levels of IL6 were significantly correlated with high OCT4/NANOG expression. IL6 stimulated an autocrine IGFI/IGFIR expression STAT3 dependently, which stimulated stemness-related properties in both the cell lines and the xenografted mouse tumors. The inhibition of IGFIR activation by either RNA interference or by treatment with the inhibitor picropodophyllin (PPP) significantly suppressed the IL6-induced stemness-related properties both in vitro and in vivo.

CONCLUSIONS

The expression of pluripotency-related genes is associated with early tumor recurrence and is regulated by IL6-induced IGF/IGFIR activation, particularly in HBV-HCC.

The purpose of this study was to investigate the roles of androgenic and estrogenic mechanisms in the stimulation of structural growth and plasma GH in male puberty. To resolve these two possible mechanisms, we compared the effect of two androgens in the treatment of constitutional delay in growth and adolescence: an aromatizable androgen, testosterone (T), and a nonaromatizable androgen, dihydrotestosterone (DHT). Nine adolescent males, Tanner stage 1 or 2, were studied before and during treatment with T enanthate (group A) or DHT heptanoate (group B). After 2.5 months of treatment, the height velocity (HV) was 12.6 +/- 2.8 cm/yr (n = 3) in group A and 8.9 +/- 1.7 cm/yr (n = 6) in group B, both within the range of peak HV for pubertal males. In group A, the integrated concentration of GH (ICGH) increased from 3.12 +/- 0.90 to 13.67 +/- 6.0 micrograms/L (P < 0.05), and plasma insulin-like growth factor-I (IGFI) increased from 126.7 +/- 2.5 to 350.3 +/- 20.3 micrograms/L (P < 0.01); plasma T increased from 0.8 +/- 0.5 to 33.8 +/- 11.0 nmol/L (P < 0.001), and the LH response to LHRH decreased from 27.6 +/- 10.7 to 5.9 +/- 2.5 IU/L (P = NS). In group B, ICGH decreased from 4.32 +/- 0.61 to 2.39 +/- 0.42 (P < 0.025), and IGF-I decreased from 218.3 +/- 39.2 to 184.0 +/- 15.8 (P = NS). Plasma T increased from 2.0 +/- 0.5 to 2.7 +/- 0.8 (P = NS), and the LH response to LHRH decreased from 45.7 +/- 14.5 to 10.7 +/- 5.8 (P < 0.05). To further evaluate the mechanism of the effect of DHT on plasma GH, seven male subjects with adolescent gynecomastia were treated with DHT heptanoate, and their responses were studied at 1 week and 3.5 months. ICGH decreased in conjunction with a decrease in the integrated T concentration (r = -0.77; P < 0.001) and to a slight degree with decreasing plasma estradiol (r = -0.39; P < 0.2). Plasma IGF-I did not show a significant change in the subjects with gynecomastia. Thus, the increase in GH at puberty in males appears to be due to an estrogen-dependent mechanism. The suppressive effect of DHT on GH secretion may be due to either suppression of estradiol production or a direct effect. Acceleration of HV into the peak pubertal range by DHT without an increase in plasma GH suggests that an increase in GH is not necessary for the pubertal growth spurt.

BACKGROUND

Adiponectin, an insulin-sensitizing adipokine, is inversely associated with adiposity and prostate cancer risk and progression. However, the role of genetic variation in the adiponectin (ADIPOQ) and receptor genes (ADIPOR1/R2) in prostate cancer is largely unknown.

METHODS

In a nested case-control study of 1,286 cases and 1,267 controls within the Physicians' Health Study, we evaluated 29 common single-nucleotide polymorphisms (SNP) in ADIPOQ (n = 13), ADIPOR1 (n = 5), and ADIPOR2 (n = 11) in relation to the risk of prostate cancer. In subgroups, we also evaluated the association of genotype and circulating adiponectin levels (n = 951) and prostate tumor expression of insulin receptor (IR) and insulin-like growth factor 1 (IGF-IR) receptor (n = 181).

RESULTS

Among the 12 tagging polymorphisms in ADIPOQ, four (rs266729, rs182052, rs822391, and rs2082940) were significantly associated (P < 0.05) with overall prostate cancer risk, with no significant difference by tumor grade or clinical stage. Two of the risk SNPs (rs266729 and rs182052) plus four other SNPs (rs16861209, rs17366568, rs3774261, and rs7639352) were also associated with plasma adiponectin levels, and three of these (rs1686109, rs17366568, and rs3774261) were also significantly associated with IR expression in prostate tumor tissue. One additional SNP was associated with IGFI-R tumor tissue expression (rs16861205). None of the 16 variants in ADIPOR1/R2 were related to cancer risk or circulating adiponectin levels.

CONCLUSIONS

Common variants in the adiponectin gene were associated with prostate cancer risk, plasma adiponectin levels, and IR or IGF-IR expression in the prostate tumor.

CONCLUSIONS

These genotype-phenotype associations support the biological relevance of adiponectin for prostate carcinogenesis, particularly in earlier stages of development.

OBJECTIVE

The insulin-like growth factor (IGF) signaling system is involved in breast cancer initiation and progression. The prognostic relevance of tumor expression patterns of IGFI-related proteins remains poorly understood. This study associates the expression of selected IGF proteins with breast tumor and patient characteristics.

METHODS

IGFI, IGFI receptor, IGF-binding protein (IGFBP)2, and IGFBP3 expression was measured in 855 primary breast carcinomas by immunohistochemistry using tissue microarrays. We investigated the association of tumor and nodal stage, grade, hormone receptor status, HER2 gene amplification, menopausal status, body mass index, and survival with IGF protein expression.

RESULTS

In contrast to IGFI, the expression of IGFI receptor, IGFBP2, and IGFBP3 was associated with estrogen receptor status. In addition, IGFBP3 was positively correlated with body mass index and premenopausal status. Importantly, IGFBP2 was an independent and positive predictor of overall survival (hazard ratio, 0.48; 95% confidence interval, 0.24-0.95; P = 0.04). There was a weak suggestion for IGFBP2 and overweight to modify each other's effect on survival.

CONCLUSIONS

According to these results, which need confirmation in larger patient series, the prognostic relevance of IGFBP2 and IGFBP3 protein expressions in breast cancer may depend on the hormonal context and body weight.

Human adult mesenchymal stem cells (hMSCs) differentiate into an osteogenic lineage if the appropriate differentiative cues, such as dexamethasone or bone morphogenetic protein 2 (BMP-2), are present. This study was undertaken to determine the role of insulin-like growth factor I (IGFI) in the regulation of early osteoblast differentiation in hMSC. Previous studies have shown that IGF-I, regulates bone formation and remodeling by participating in the differentiation of mature cells of osteoblast lineage. We hypothesized that IGF-I exerted its effects early, but the effects were too subtle to be detected. Therefore, engineered hMSCs to produce IGF-I via adenoviral transfection and used quantitative real-time PCR (qPCR) to assess marker gene expression. Here we show that IGF-I up-regulates Type I collagen, Runx2, and alkaline phosphatase (Alp) gene expression in hMSCs, genes indicative of early osteogenic differentiation. We also observed mineral deposition in the absence of dexamethasone (Dex) in hMSC cultures treated with recombinant human BMP-2 after transduction with Ad-IGF-I. In conclusion Igf-I transduction up-regulated markers of osteoblastic differentiation and in conjunction with recombinant BMP-2-induced matrix mineralization independently of Dex (see Salasznyk et al., Stem Cells Dev 14(6):608-620, 2005, this issue).

The large offspring syndrome (LOS) is observed in bovine and ovine offspring following transfer of in vitro-produced (IVP) or cloned embryos and is characterized by a multitude of pathologic changes, of which extended gestation length and increased birthweight are predominant features. In the present study, we used bovine blastocysts to analyze cellular parameters, i.e., the number of cells in Day 7 blastocysts and the size of Day 12 elongating blastocysts, and molecular parameters, i.e., the relative abundance of developmentally important genes: glucose transporter (Glut) 1, Glut-2, Glut-3, Glut-4, heat shock protein (Hsp) 70.1, Cu/Zn-superoxide dismutase (SOD), histone H4.1, basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF) I receptor (R), and IGFII-R. Some blastocysts were produced by in vitro maturation and fertilization followed by in vitro culture in synthetic oviduct fluid medium supplemented with BSA or human serum or by in vivo culture in the sheep oviduct. Other blastocysts were derived in vivo from the uterine horns of superovulated donors. The findings made in the early embryos were related to a representative number of calves obtained from each production system and from artificial insemination (AI). In vitro culture of bovine embryos in the presence of high concentrations of serum or BSA significantly increased the number of cells in Day 7 blastocysts, the size of blastocysts on Day 12, and the relative abundance of the transcripts for Hsp70.1, Cu/Zn-SOD, Glut-3, Glut-4, bFGF, and IGFI-R when compared with embryos from the in vivo production groups. Birthweights of calves derived from IVP embryos were significantly higher than those of calves derived from sheep oviduct culture, superovulation, or AI. The results support the hypothesis that persistence of early deviations in development is causally involved in the incidence of LOS, in particular in increased birthweights. The cellular and molecular parameters analyzed in this study can be considered early markers of LOS in cattle.

Soy isoflavones are hypothesized to exert hormonal effects in women and thus may play a role in bone metabolism throughout life. In 2 randomized, cross-over studies, 14 pre- and 17 postmenopausal women were given 3 soy protein isolates containing different amounts of isoflavones [control, 0.13; low isoflavone (low-iso), 1.00; and high-iso, 2.01 mg/kg body wt/day, averaging 8, 65, and 130 mg/day, respectively], for over 3 months each. Food records, blood samples, and 24-h urine collections were obtained throughout the studies. The endpoints evaluated included plasma or serum concentrations of bone-specific alkaline phosphatase, osteocalcin, insulin-like growth factor-I (IGFI), IGF binding protein-3 (IGFBP3), and urine concentrations of deoxypyridinoline cross-links and carboxy-terminal telopeptide of type I collagen. In premenopausal women, IGFI and IGFBP3 concentrations were increased by the low-iso diet, and deoxypyridinoline cross-links was increased by both the low- and high-iso diets during certain phases of the menstrual cycle. In postmenopausal women, bone-specific alkaline phosphatase was decreased by both the low- and high-iso diets, and there were trends toward decreased osteocalcin, IGFI, and IGFBP3 concentrations with increasing isoflavone consumption. Although soy isoflavones do affect markers of bone turnover, the changes observed were of small magnitude and not likely to be clinically relevant. These data do not support the hypothesis that dietary isoflavones per se exert beneficial effects on bone turnover in women.

Using subcloning and manipulations of culture conditions we have isolated from the mouse myogenic cell line C2 a variant cell line that we named inducible. Unlike the progenitor cells that are referred to as permissive, inducible myoblasts differentiate poorly in Dulbecco modified Eagle medium plus fetal calf serum (FCS) and require the presence of insulin at a high concentration (1.6 10(-6) M) or insulin-like growth factor I (IGFI) at a lower concentration (2.5 10(-8) M) to differentiate. Permissive and inducible myoblasts fail to differentiate when grown in MCDB202 medium plus 20% FCS, even after a prolonged arrest in G1 phase. This shows that an arrest in G1 is in itself insufficient to trigger terminal differentiation. Both cell types also exhibit distinct patterns of accumulation of muscle mRNAs corresponding to sarcomeric actins and myosin light chain MLC1A. The possibility that these two cell lines might represent two different stages of the progression of myoblasts toward terminal differentiation is discussed.

Protein tyrosine kinases have been implicated in tumor initiation and progression. Here we used Northern blotting to study expression of their genes in cultured normal melanocytes and 19 melanoma cell lines from different stages of tumor progression. We detected transcripts for 2 cytoplasmic (ABL and FES) and 6 receptor (ECK, ERB-B2, FGF-R4, IGFI-R, KDR and TIE) kinases but not for receptors RET or TRK-A. Genes for ECK, FGF-R4 and TIE were expressed ectopically in melanomas (not in normal melanocytes). Similarly, ECK protein was detected by immunoblotting in metastatic melanomas but not in normal melanocytes. ECK mRNA levels tended to increase again during late melanoma progression. ECK and TIE mRNAs were also detected in highly metastatic variant cells but not in the corresponding poorly metastatic parental lines. Conversely, FES and KDR gene expression was lost in most advanced primary and metastatic melanomas. These findings suggest positive and negative roles for specific tyrosine kinases during progression.

Physiological angiogenesis is essential for development, homeostasis and tissue repair but pathological neovascularization is a major feature of tumours, rheumatoid arthritis and ocular complications. Studies over the last decade have identified gamma-secretase, a presenilin-dependent protease, as a key regulator of angiogenesis through: (i) regulated intramembrane proteolysis and transmembrane cleavage of receptors (e.g. VEGFR-1, Notch, ErbB-4, IGFI-R) followed by translocation of the intracellular domain to the nucleus, (ii) translocation of full length membrane-bound receptors to the nucleus (VEGFR-1), (iii) phosphorylation of membrane bound proteins (VEGFR-1 and ErbB-4), (iv) modulation of adherens junctions (cadherin) and regulation of permeability and (v) cleavage of amyloid precursor protein to amyloid-? which is able to regulate the angiogenic process. The gamma-secretase-induced translocation of receptors to the nucleus provides an alternative intracellular signalling pathway, which acts as a potent regulator of transcription. gamma-secretase is a complex composed of four different integral proteins (presenilin, nicastrin, Aph-1 and Pen-2), which determine the stability, substrate binding, substrate specificity and proteolytic activity of gamma-secretase. This seeming complexity allows numerous possibilities for the development of targeted gamma-secretase agonists/antagonists, which can specifically regulate the angiogenic process. This review will consider the structure and function of gamma-secretase, the growing evidence for its role in angiogenesis and the substrates involved, gamma-secretase as a therapeutic target and future challenges in this area.

Despite the findings that β1 integrins play a vital role in the regulation of cell proliferation and survival, the mechanisms through which they operate and lead to cancer progression remain elusive. Previously, our laboratory has shown that β(1A) integrins support insulin-like growth factor 1 (IGFI)-mediated mitogenic and transforming activities. Here, we report that β(1A) integrins regulate basal levels of IGF-IR, although they are not critical for maintaining cancer cell morphology. Upon transfection of β(1A) siRNA and consequent downregulation of IGF-IR, we show inhibition of anchorage-independent growth of prostate cancer cells, a function which is dependent on IGF-IR expression. In addition, we demonstrate that IGFI-mediated activation of androgen receptor (AR), known to occur in prostate cancer cells, requires expression of β(1A) integrins as evaluated by luciferase reporter assays and immunoblotting analysis. Since β(1A) integrin levels are increased by R1881 or dihydrotestosterone (DHT), our results imply that β(1A) integrins support an androgen-enhanced feedback loop that regulates the expression of IGF-IR. β(1A) integrins also regulate inducible levels of IGF-IR in cells stimulated by androgen or by a combination of androgen and IGFI, as evaluated by flow cytometric analysis and immunoblotting. Furthermore, upon transfection of β(1A) siRNA and consequent downregulation of IGF-IR, neither activation of AKT, an effector of IGF-IR, nor AR levels are affected. We conclude that β(1A) integrin expression is critical for maintaining the regulatory crosstalk between IGF-IR and AR.